Many cancers are known to have markedly different rates of incidence, and very different outcomes, in males and females. Among these are brain tumors, the most common of them being glioblastomas (GBM). These are the most aggressive tumors of the brain and occur at 60% higher rates in males, whether human or not. Now a startling new study published on the preprint server bioRxiv* in November 2020 tracks down these differences to a gene called Brd4, a transcriptome-wide regulator of gene expression.
Recently, it was recognized that disease susceptibility and clinical phenotype are determined not only by pathogenic and environmental factors but also by the sex of the patient. This is also true for lifestyle diseases, including metabolic conditions like cardiovascular and neurologic disease and cancer.
The new study explored differences in GBM biology in males and females to better understand underlying factors and pathways that drive the risk and course of tumors. Earlier, the researchers found that GBM cells in male mice were more vulnerable to cancerous change-inducing events and the effects of chemotherapy. Half of these differences were confirmed in human GBM.
A later study showed that differences in survival in treated human GBM patients were due to the underlying transcriptional programs modulated by sex-specific epigenetic changes, just as with normal sexual differentiation.
The BET family of proteins is involved in regulating transcription by epigenetic reading and work together with target genes to which they recruit specific transcriptional complexes. Brd4 is a BET protein that reads acetylated histones H3 and H4 throughout the entire cell cycle and specifies its cell identity.
The enhancers bound to it may therefore be responsible for the fundamental sex-dependent differences in GBM. Moreover, Brd4 inhibition is being increasingly targeted by drugs for epigenetic modulation of growth in many cancers. It is found to be un-regulated in many cancers. It enhances many processes such as epithelial-to-mesenchymal transition, conversion to a stem cell-like profile, and pluripotency.
The current study shows that the sex differences in the tumor phenotype vary with the effect of the enhancer regulatory molecule bound to Brd4 on the course of stem cell-like differentiation in male and female GBM cells. Inhibition of Brd4 by genetic and pharmacological factors also varies in males and females, both in vivo and in vitro. Thus, GBM cells in males are less likely to form new clones and tumors are less likely to grow, following Brd4 inhibition. The opposite occurs in female cells and tumors.
The researchers found that Brd4 binding sites were at regions with a high density of H3 acetylation. Of the Brd4-bound enhancers, a fifth (around 2,800) bound more Brd4 in males, while the same proportion bound more of the protein in female GBM cells. Of the former, 0.13% were on the Y chromosome and 3.11% on the X chromosome. Of the latter, 4.29% were located on the X chromosome. The researcher's comment, “The observed differences in Brd4-bound enhancers are not simply due to differential Brd4 enhancer enrichment on sex chromosomes.” In fact, this is the first time that male- and female-biased Brd4-bound enhancer use has been shown in any type of cell.
The researchers also found that Brd4-bound genes were regulated differently by males and females; in around 1,300 genes, about 52% were expressed more often in males. The pathways most often regulated by these genes involve core cancer pathways, which are therefore differently regulated in the sexes. Not only are the transcriptional programs leading to tumor formation different between males and females, but their binding by Brd4-enhancers is also dependent on the sex.
The response to small molecule inhibitors of BET proteins in male GBM cells was at reduced clonogenic cell frequency, but in females, it was increased. That is, the sex differences were reduced by Brd4 inhibition because of the responses which occurred in opposite directions. The drugs reduced tumor growth in males but increased it in females.
The authors point out, “These results demonstrate for the first time that the sex differences in the tumorigenic phenotype we observe in our murine GBM cells are mediated by differential Brd4-bound enhancers and that the response to BET inhibition is sex-dependent.”
Similar results were later obtained with human GBM cells. Thus, not only the biology of these tumors but their response to therapeutic drugs is sex-dependent.
Earlier studies showed that women with ER-positive breast or endometrial cancer had poor survival odds if Brd4 expression was low, but in men with prostate cancer, the same marker indicated better survival.
Transcription factors concerned with oncogenesis and stem cell-like development were enriched at male-biased Brd4-bound enhancers, but at female-biased Brd4-bound enhancers were bound to tumor suppressor genes such as p53. Thus, this gene is apparently able to promote tumor formation in male GBM but to suppress it in female GBD.
Further study will be needed to understand the master transcriptional regulators which bind to Brd4, directly or indirectly, to determine where it occurs in the genome. However, the study showed that Brd4 is found in the same location as Myc and p53, in male and female GBM cells, respectively.
The authors conclude: “We have identified sex-biased Brd4-regulated genes and pathways, which could translate into new and promising therapeutic targets to enhance survival for all GBM patients and potentially other cancers that exhibit substantial sex differences in incidence or outcome.”